Mycobacterium tuberculosis is one of the most important opportunistic pathogens of HIV-infected individuals. Current treatments for tuberculosis are being threatened by the rapid emergence of drug resistance. Our research program has worked to define the mechanisms of action of a leading anti-tuberculosis drug isoniazid (INH) and an important second line drug ethionamide (ETH), with the goal of enabling rational drug design. Using a combination of genetics, biochemistry, X-ray crystallography, electron microscopic and gene analysis approaches, we discovered a common target to be an enoyl reductase of the Fatty Acid Synthase (FAS) type II system responsible for mycolic acid synthesis. The three dimensional structure of InhA was determined, and its enzymatic activity was unexpectedly found be in inhibited by an INH-NAD adduct that bound to the InhA NADH binding pocket. We have also identified a set of contiguous genes iniB, iniA, and iniC, that confer tolerance to INH in mycobacteria overexpressing these genes. We have established InhA as an excellent drug target by demonstrating that InhA-thermal inactivation of a temperature-sensitive mutation in inhA leads to lysis of the mycobacterial cell. Moreover, we have identified novel compounds that inhibit InhA and possess anti-mycobacterial activity. Despite these accomplishments, additional questions remain concerning the molecular events that lead to INH-induced cell lysis, and the mechanisms that confer resistance to this phenomenon. Here we propose to continue our multi-disciplinary approach to study novel resistance mechanisms, including one that is caused by defects in NADH dehydrogenase. We will also focus on defining the molecular events that lead to mycobacterial cell lysis by comparing and contrasting three different mutant strains that cause lysis following inhibition of three different metabolic pathways. Finally, we will characterize the tolerance mediated by the IniA and IniB proteins, and define the mechanisms by which mycobacteria prevent lysis. The knowledge gained by this work will lead to the identification of novel drug targets, strategies to overcome tolerance, and more effective treatments for tuberculosis.